MICROBIAL STOICHIOMETRY

(CELLS AND PRODUCT FORMATION)

Definition : Science related with

•Quantitative compositions of
chemicals, and
•Quantitative conversion of chemical
reactions.
Objective : Understand quantitave flows of
materials in biological system.
Stoichiometry

• Provides information on fundamental

constraints
– Substrate conversion to product
– Cell mass from substrate
Useful for :

• Media formulation
• Determining materials needs and their prices.
• Calculating yield of cells, products, and by-products or
waste.

 Size of equipment and cost.
 Operational cost
 Heat transfer to/from process.
Yields and yield coefficients
• Mass based = “kg” of this from “kg” of that
• Y (output / input)
• Y x/s
• Y p/s
• Y ATP/O2

• Ymx/s maximal yield of cell mass from

substrate
•Yield of cell mass from substrate
Y x/s

Bacterial
dry cell weight Slope = dX/dS
[mg/L] 7 (mg/L) / (g/L)

Glucose [g/L]
 Aerobic
Yx/s=58 mg/mol

Bacterial
dry cell weight
[g/L]
Anaerobic
Yx/s=22 mg/mol

Glucose [mM]
Cell composition
Dry weight vs. wet weight
70% of the composition is water

CHxOyNz
Dry weight consists of:
Element E. coli Yeast

C 50% 50%
O 20% 34%
N 14% 8%
H 8% 6%
P 3% 1%
S 1% <1%
K 1% <1%
Na 1% <1%
Others <1% <1%
• In a very simplistic interpretation of
metabolism, the following applies:

– Cells + medium + O2 (sometimes) → more cells +

product + CO2 + H2O

• Medium contains sugars, amino acids,

cofactors and the elements in the previous
table.
• In a very simplistic interpretation of
metabolism, the following applies:

– Cells + medium + O2 (sometimes) → more cells +

product + CO2 + H2O

• Medium contains sugars, amino acids,

cofactors and the elements in the previous
table.
Stoichiometric calculations
• Based on 1 mole of C in the input
• CHmOn + a O2 + b NH3 →

• c CHaObNd + dH2O + eCO2

• This is normalized to 1 mole of C.
Could also be normalized to 1 mole of the C source compound

• Perform elemental balances to determine the

unknown values of the cofactors
Elemental Balances
1. Simple Conversion
Simple conversion of C and N sources into
Cell, H2O and CO2 without extracellular
product.
CHmOn + a O2 + b NH3  c CHaObNd +
d H2O + e CO2
CHmOn = 1 mole of carbohydrate
CHaObNd = 1 mole of cellular material.
(typical cell composition CH1.8O0.5N0.2)
ELEMENTAL BALAN CES
Elemental balances of C,H,O,N are as follows:
C :1=c+e
H : m + 3b = ca + 2d
O : n + 2a = cb + d + 2e
N : b = cd
Respiratory quotient
RQ = e/a
Example

• C6H12O6 + a O2 + b NH3 →

• c C4.4H7.3O1.2N0.86 + dH2O + eCO2

• 2/3 of the glucose C goes to biomass

• What are the stoichiometric coefficients,
and Yx/s, Yx/O2? MWglucose = 180
MWcell = 91.34
MWoxygen = 32
MWammonia = 17
 Elemental Balance on C (C balance)
Amount of C (mass) in 1 mole substrate = 6 x
12 = 72 g
Amount of C converted into biomass = 2/3(72 )
= 48 g
therefore,
48 = 4.4 (c)(12)
c = 0.909
Amount of C converted into CO2, is 72 – 48 = 24
g
therefore,
24 =12e
e=2
• N balance
14b = 0.86 (c)(14)
b = 0.782

 H Balance
12 + 3b = 7.3c +2d
d = 3.854

 O balance O
6(16) +12 (a)(16) = 1.2 (c)(16) + 16d + 2
(e)(16)
a = 1.473
• Theoretical Yield
Yx/s = (0.909)(91.34)/180
= 0.461 g dry cell/g substrate

Yx/O2 = (0.909)(91.34)/(1.473)(32)
= 1.76 g dry cell/ g O2
• On the previous elemental balances, there
are 5 equations for 5 unknown variables. If
RQ can be measured, the equations can be
solved to get the stoichiometric coefficients.

For more complex reactions, involving

extracellular product formation, additional
parameters are needed to solve the
equations.
Respiratory quotient
• RQ = YCO2/O2

• Molar basis
– Moles of CO2 produced from moles of O2

• Provides information on the metabolic state of the cell

• A high RQ means that much CO2 is produced and hence

the metabolism is operating at high efficiency
Aerobic metabolism

• CHmOn + a O2 + b NH3 →

• c CHaObNd + d CHxOyNz + eH2O + fCO2

• RQ = ?
Generalized growth reaction
• C6H12O6 + a NH3 + b O2 → a CH1.8O0.5N0.2 +
 b CHxOyNz + gCO2 + dH2O

• Normalized to 1 mole of carbon source compound

• Where a, b, a, b, g, d, x, y, z depend on the type of cell
involved.
• a, b, a, b, g, d, are stoichiometric coefficients
• When little info is available about cell composition, use
an approximated cell composition of
CH1.8O0.5N0.2
• This yields a MW of a cell ~ 24.6
Generalized growth reaction

C6H12O6 + a NH3 + b O2 → a CH1.8O0.5N0.2 +

b CHxOyNz + gCO2 + dH2O

24.6a
Yx s 
180
g of cells from g of glucose
Lack of information

• Unfortunately, the elemental balances

often do not provide enough information
to completely solve for the stoichiometric
coefficients.
Degree of reduction

• Electron balance
g = # of available electrons / g of atomic C
g Or, this can be described as:
g = # of available electrons / # of C’s

• Provides another independent equation

Degree of reduction
• C=4 • CO2 = +4 (C) + -2 (O) = 0
• H=1
• N = -3 • C6H12O6 = 6(4) + 12(1) + 6(-2) = 24
– g = 24 / 6 (# carbon atoms) = 4
• O = -2
• P=5 • C2H5OH = 2(4) + 6(1) + (-2) = 12
• S=6 – g = 12 / 2 (# carbon atoms) = 6
Degree of Reduction of
Substrate
Degree of reduction is calculated as follows :

Methane (CH4) : 1(4) + 4(1) = 8, g = 8/1 = 8

Glucose (C6H12O6) : 6(4) + 12(1) + 6(-2) =24, g =
24/6 = 4
Ethanol (C2H5OH) : 2(4) + 6(1) + 1(-2) = 12, g =
12/2 = 6
Example : Production of extracellular product
aerobically

CHmOn + a O2 + b NH3  c CHaObNd + d CHxOyNz +

e H2O + f CO2

Degree of reduction for substrate, cell biomass and

product are as follows;
gs = 4 + m - 2n
gb = 4 + a - 2 b - 3 d
gp = 4 + x - 2 y - 3 z
Degree of reduction for CO2, H2O and NH3 = 0
The equation can be solved using elemental
balances (C, H, O and N), degree of reduction,
energy balances, and total mass balances.

Since the amount of water formed and used in the

reaction are difficult to determine, and water is in
excess, so the H and O balances are difficult to
use. Therefore, C and N balances, and degree of
reduction are used..

Degree of reduction
C : 1 =c + d + f
N : b = cd + dz
g : gs - 4a = c gb + d gp
TEORETICAL PREDICTION OF YIELD

•
• Example

Estimate theoretical yield coefficient for cell and

product formation in the ethanol fermentation by S.
cerevisiae as described in the following reaction:

C6H12O6  2 C2H5OH + 2 CO2

• Answer:

For complete conversion of glucose into ethanol maximum

product yield is :
Yp/s = 2 x 46/180 = 0.51 g ethanol/g glucose
•
While maximum yield for CO2:
YCO2/s = 2 x 44/180 = 0.49 g CO2/g glucose.

Practically, product yield is about 90 – 95% from the theoretical

maximum yield since some glucose is converted into cell
biomass and by product such as glycerol and acetic acid.